In recent years, various biosensors utilizing a specific catalytic action possessed by enzyme have been developed and in particular, it has been attempted to apply biosensors to the clinical field, and biosensors which can provide rapid assay with good accuracy have been desired.
Taking a glucose sensor as an example, diabetes has markedly increased nowadays and for measurement and control of blood glucose concentration, it takes a very long time, since blood is centrifuged and plasma is provided for the measurement as is conventionally done. Thus, a sensor which can make measurement with whole blood is required. As a handy type, there is a stick-like support having provided thereon a carrier containing an enzyme capable of reacting only with glucose and a dye which changes color upon enzyme reaction or by the product of the enzyme reaction, like a test sheet used for inspection of urine. The stick takes the system that blood is dropped onto the carrier and after a definite period of time, a change of the dye is visually or optically determined. However, interference is serious because of colored matters in blood, resulting in poor accuracy.
Now, a multilayer type analysis carrier as shown in FIG. 1 is proposed (Japanese Utility Model Application Laid-Open No. 54-178495). The carrier has the construction comprising a transparent support 51 having provided thereon, in order, a reagent layer 52, a developing layer 53, a waterproofing layer 54 and a filtering layer 55. The measurement takes the following system: when a blood sample is dropped from the upside, solid components in blood such as red blood cells, platelets, etc. are removed by the filtering layer 55, the blood uniformly permeates into the developing layer 53 through a hole 56 in the waterproofing layer and a reaction proceeds in the reagent layer 52. After completion of the reaction, a light is irradiated from the arrow direction through the transparent support 51, whereby a substrate concentration is determined by colorimetry. The system takes a complicated construction as compared to the conventional handy stick-like carrier but its accuracy has improved because blood cells are removed, etc. However, it takes a long time for the permeation of blood and the reaction so that the waterproofing layer 54 that prevents drying of the sample is required. In addition, incubation at a high temperature is required for accelerating the reaction. Thus, the system involves problems that apparatuses and carriers become complicated.
On the other hand, as the system for quantitative assay of a specific component in a sample such as blood, etc. from the living body with high accuracy without performing operations such as dilution, agitation, etc. of the sample solution, a biosensor as shown in FIG. 2 has been proposed (for example, Japanese Patent Application Laid-Open No. 59-166852). The biosensor comprises an insulating base plate 63 having embedded therein a working electrode 64 and a counter electrode 65 made of platinum, etc., having leads 61 and 62, respectively, and the exposed areas of these electrodes are covered with a porous material 66 having carried thereon an oxidoreductase and an electron acceptor. When a sample solution is dropped onto the porous material, the oxidoreductase and the electron acceptor are dissolved in the sample solution, whereby an enzyme reaction proceeds with a substrate in the sample solution and the electron acceptor is reduced. After completion of the reaction, the reduced electron acceptor is electrochemically oxidized and a substrate concentration in the sample is determined from a current level for the oxidation obtained in this case.
In such a construction, however, the electrodes require operations such as washing, etc., while the porous material can be exchanged for every assay thereby to readily provide for measurement. On the other hand, if it is possible to dispose the apparatus including the electrode system for every measurement, operations for the measurement become extremely simple but from aspects of electrode materials such as platinum, etc., construction and the like, the apparatus is very expensive unavoidably. For the construction of platinum electrodes, the sputtering method or the deposition method can also be used but production costs increase.
As a disposable system including the electrode system, a biosensor described in Japanese Patent Application Laid-Open No. Hei. 01-291153 has been proposed.
As shown in FIGS. 3 and 4 in this bio-sensor electric leads 2 and 3, electrodes consisting of a working electrode 4 and a counter electrode 5, and an insulative layer 6 are formed on an insulative base plate 1 by means of screen printing and the like, and an enzyme reaction layer 14 consisting of a hydrophilic polymer, an oxidoreductase and an electron acceptor is formed on the electrodes, which are integrated with a spacer 10 and a cover 11. When a sample liquid is introduced onto the enzyme reaction layer 14 from a sample feed 12, the air of the reaction layer 14 is removed from the air outlet 13, and the reaction layer 14 is rapidly filled with the sample liquid. The reaction layer 14 is dissolved with the sample liquid, the enzyme reaction progresses between oxidoreductase and the substrate in the sample liquid, and the electron acceptor is reduced. After the enzyme reaction is completed, the reduced electron acceptor is electrochemically oxidized to determine the substrate concentration in the sample liquid according to the value of the electric current at this oxidation.
It was difficult to produce a biosensor having an even quality in the above constitution, because the surface state of the electrode system produced according to a method such as screen printing which is usually adopted to produce a disposable biosensor economically becomes finely varied to give an uneven sensor response. Additionally, according to the above method the wettability of surface of the base plate containing the electrodes is so much worse that a solution containing an enzyme is repelled on the electrodes, when coated or spread on the electrodes, so as to hardly form the reaction layer often.
Therefore, as a method of preparing sensors for measuring a specific component in a biological liquid sample such as blood or the like in a simple and rapid way with high accuracy, a method is desired by which an even reaction layer can be easily formed. Further, it is desired that the biosensor has a good storage stability.